PAIP2 Antibody

What is PAIP2 and why is it important in cellular function?

PAIP2 (poly(A) binding protein interacting protein 2) is a cytoplasmic protein that functions as a translation repressor by interacting with poly(A) binding protein cytoplasmic 1 (PABPC1). Its primary role involves regulating translation initiation of poly(A)-containing mRNAs through its interaction with PABPC1, which is an essential translational initiation factor . The protein contains conserved PABPC1-interacting motifs (PAM1 and PAM2) that are crucial for its binding to PABPC1 . Additionally, PAIP2 appears to play a role in mRNA deadenylation, potentially by promoting the dissociation of PABPC1 from shortened mRNA . This function positions PAIP2 as an important factor in post-transcriptional gene regulation mechanisms.

What are the basic properties of human PAIP2 protein?

Human PAIP2 protein consists of 127 amino acid residues with a calculated molecular weight of 15 kDa, although it is commonly observed at 26 kDa in experimental conditions . It is primarily localized in the cytoplasm and is expressed at varying levels across different tissues, with highest expression in testis and significant presence in brain, cervix, lung, ovary, placenta, adipose tissue, thymus, and thyroid . PAIP2 is a member of the PAIP2 protein family and is also known by several synonyms including polyadenylate-binding protein-interacting protein 2, PABC1-interacting protein 2, and PAIP2A . The protein is subject to post-translational modifications, particularly ubiquitination, which affects its stability and function .

What applications are PAIP2 antibodies commonly used for?

PAIP2 antibodies are utilized in multiple experimental applications to detect and study PAIP2 protein. According to available data, these antibodies have been validated for:

Researchers should note that optimal antibody dilutions may vary depending on specific experimental conditions and sample types . It is recommended to perform titration experiments to determine optimal antibody concentrations for each specific research application and sample type.

How should I optimize Western blot protocols for PAIP2 detection?

When optimizing Western blot protocols for PAIP2 detection, several key considerations should be addressed. First, recognize that while the calculated molecular weight of PAIP2 is 15 kDa, it is typically observed at approximately 26 kDa on SDS-PAGE gels . This discrepancy may be due to post-translational modifications or the protein's structural properties affecting its migration pattern.

For optimal results:

• Sample preparation: Use fresh cells or tissues, and include protease inhibitors in lysis buffers to prevent degradation of PAIP2.

• Gel percentage: Use 12-15% polyacrylamide gels for better resolution of PAIP2.

• Transfer conditions: Optimize transfer time and voltage for proteins in this molecular weight range.

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1 hour at room temperature.

• Primary antibody: Start with a 1:500 dilution of PAIP2 antibody and incubate overnight at 4°C .

• Washing: Perform at least 3-5 washes with TBST to reduce background.

• Secondary antibody: Use HRP-conjugated anti-rabbit IgG (for rabbit-derived PAIP2 antibodies) at 1:5000-1:10000 dilution.

• Positive controls: Include lysates from A431, HeLa, or Jurkat cells, which are known to express detectable levels of PAIP2 .

If troubleshooting is necessary, consider adjusting antibody concentration, incubation time, or detection method sensitivity.

What are the critical considerations for immunofluorescence experiments using PAIP2 antibody?

For successful immunofluorescence (IF) or immunocytochemistry (ICC) experiments with PAIP2 antibody, researchers should carefully consider:

• Fixation method: Paraformaldehyde (4%) is commonly used, but methanol fixation may better preserve certain epitopes. Test both to determine optimal conditions.

• Permeabilization: Use 0.1-0.5% Triton X-100 in PBS to ensure antibody access to cytoplasmic PAIP2.

• Blocking: Incubate with 1-5% BSA or normal serum from the species of the secondary antibody for 30-60 minutes to reduce non-specific binding.

• Antibody dilution: Begin with a 1:100 dilution for PAIP2 antibody and adjust as needed based on signal intensity and background .

• Co-staining considerations: When performing co-localization studies, ensure compatibility of primary antibodies (different host species) and secondary antibodies (minimal cross-reactivity).

• Controls: Include both positive controls (HeLa cells have been validated) and negative controls (primary antibody omission, isotype controls).

• PAIP2 localization pattern: Expect predominantly cytoplasmic staining, consistent with its known subcellular localization .

For quantitative analysis, maintain consistent acquisition parameters across all samples and consider using Z-stack imaging to capture the full cytoplasmic distribution of PAIP2.

How can PAIP2 antibodies be used to investigate mRNA translation regulation mechanisms?

PAIP2 antibodies can be instrumental in investigating mRNA translation regulation through several sophisticated experimental approaches:

• RNA immunoprecipitation (RIP): Using PAIP2 antibodies for RIP allows researchers to identify mRNAs associated with PAIP2 in vivo. This approach can help determine which transcripts are preferentially regulated by PAIP2-mediated repression.

• Polysome profiling: Combining polysome fractionation with PAIP2 immunoblotting can reveal the association of PAIP2 with different translation states and ribosomal fractions, providing insights into how PAIP2 affects the translation efficiency of specific mRNAs.

• Proximity ligation assays (PLA): This technique can visualize and quantify the interaction between PAIP2 and PABPC1 or other translation factors in situ, offering spatial information about these interactions within the cell.

• PABPC1 displacement studies: PAIP2 antibodies can be used to investigate how PAIP2 affects PABPC1 binding to poly(A) tails during deadenylation. Research suggests that PAIP2 may enhance deadenylation by promoting dissociation of PABPC1 from shortened mRNAs .

• Protein complex immunoprecipitation: PAIP2 antibodies can be used to immunoprecipitate PAIP2 and its interacting partners, allowing for the identification of novel protein complexes involved in translation regulation.

When designing these experiments, it's critical to verify that the PAIP2 antibody being used does not interfere with the protein-protein or protein-RNA interactions under investigation.

What is the significance of PAIP2 in cancer research and how can antibodies help elucidate its role?

PAIP2 has emerging significance in cancer research, particularly in breast cancer, where it shows potential as a diagnostic and prognostic biomarker . PAIP2 antibodies can facilitate several critical investigations in cancer research:

When interpreting PAIP2 expression in cancer contexts, researchers should consider its relationship with specific immune cell populations, as PAIP2 has been found to positively correlate with resting mast cells and M2 macrophages while negatively correlating with follicular helper T cells and CD8+ T cells .

How does the PAIP2-PABPC1 interaction influence protein stability, and how can this be investigated?

The interaction between PAIP2 and PABPC1 appears to be critical for PAIP2 stability, as disruption of this interaction can lead to decreased PAIP2 protein levels . This relationship can be investigated using several methodological approaches:

• PAM2 competition assays: Overexpression of PAM2 motifs can competitively inhibit PAIP2-PABPC1 interaction. Research has shown that cells expressing PAM2-GFP fusion proteins display decreased PAIP2 protein levels compared to controls .

• Mutational analysis: The F118A mutation in PAIP2's PAM2 motif disrupts binding to the MLLE domain of PABPC1. Comparing the stability of wild-type versus mutant PAIP2 can reveal the importance of this interaction for protein stability .

• Co-immunoprecipitation (Co-IP): Using PAIP2 antibodies for Co-IP can isolate PAIP2-PABPC1 complexes and assess how various conditions affect complex formation. Adding recombinant GST-super-PAM2 proteins to lysates can test the requirement for PAM2/MLLE interaction .

• Pulse-chase experiments: These can determine the half-life of PAIP2 under conditions where PABPC1 interaction is either preserved or disrupted, revealing the quantitative impact of this interaction on PAIP2 stability.

• Proteasome inhibitor studies: Treatment with proteasome inhibitors while modulating PAIP2-PABPC1 interaction can determine whether degradation is proteasome-dependent or independent .

Research has revealed that formation of the Paip2/PABPC1 complex protects PAIP2 from proteasome-independent degradation , highlighting an important post-translational regulatory mechanism for this protein.

How should researchers address the molecular weight discrepancy between calculated and observed PAIP2 size?

The discrepancy between PAIP2's calculated molecular weight (15 kDa) and its commonly observed size on SDS-PAGE (26 kDa) represents a technical challenge that researchers should be prepared to address. This phenomenon is not uncommon in protein biochemistry and can be approached methodically:

• Verification strategies:

  • Use multiple PAIP2 antibodies targeting different epitopes to confirm band identity

  • Include positive controls of known PAIP2-expressing cells (A431, HeLa, Jurkat cells)

  • Perform knockdown or knockout validation to confirm band specificity

  • Consider using recombinant PAIP2 protein as a size reference

• Technical explanations for the discrepancy:

  • Post-translational modifications such as ubiquitination can significantly increase apparent molecular weight

  • The highly charged nature of some proteins can affect SDS binding and alter migration patterns

  • Structural features like protein domains with unusual amino acid composition may affect electrophoretic mobility

• Experimental adaptations:

  • Use gradient gels (4-20%) to better resolve and identify PAIP2

  • Consider alternative electrophoresis buffer systems that may improve size resolution

  • Document both expected (15 kDa) and observed (26 kDa) sizes in experimental reports

  • For precise size determination, consider mass spectrometry analysis of immunoprecipitated PAIP2

When publishing results, clearly indicate both the calculated and observed molecular weights to avoid confusion in the field and facilitate cross-study comparisons.

What controls should be implemented when using PAIP2 antibodies for functional studies?

Implementing rigorous controls is essential when using PAIP2 antibodies for functional studies to ensure data validity and reproducibility:

• Antibody validation controls:

  • Positive tissue/cell controls: Include samples known to express PAIP2 (A431, HeLa, Jurkat cells, mouse lung tissue)

  • Negative controls: Use tissues or cells with minimal PAIP2 expression or PAIP2 knockdown/knockout samples

  • Peptide competition assay: Pre-incubate antibody with immunizing peptide to confirm specificity

  • Compare results from multiple PAIP2 antibodies targeting different epitopes

• Experimental technique-specific controls:

  • For immunoprecipitation: Include IgG control, input sample, and flow-through fractions

  • For immunofluorescence: Include secondary-only controls and isotype controls

  • For Western blot: Use loading controls (β-actin, GAPDH) and molecular weight markers

• Functional assay controls:

  • Include both gain-of-function (PAIP2 overexpression) and loss-of-function (PAIP2 knockdown) conditions

  • Use PAM2 mutant (F118A) as a control for PABPC1 interaction-dependent effects

  • Implement rescue experiments to confirm specificity of observed phenotypes

• Biological relevance controls:

  • Verify results across multiple cell lines/tissue types

  • Validate key findings with alternative methodologies not dependent on antibodies

  • Correlate antibody-based results with mRNA expression data

When publishing research utilizing PAIP2 antibodies, comprehensively document all controls and antibody validation steps to strengthen the reliability of the findings.

How can PAIP2 antibodies contribute to understanding the role of PAIP2 in immunotherapy response prediction?

PAIP2 antibodies can significantly advance our understanding of PAIP2's role in predicting immunotherapy responses through several innovative approaches:

• Patient stratification studies: Immunohistochemistry using PAIP2 antibodies can help stratify cancer patients based on PAIP2 expression levels. Research indicates that PAIP2 expression is negatively correlated with tumor mutational burden (TMB) and Immunophenoscore (IPS), suggesting that patients with low PAIP2 expression may respond better to immune checkpoint inhibitors .

• Multiplex immune profiling: PAIP2 antibodies can be incorporated into multiplex immunofluorescence panels to simultaneously evaluate PAIP2 expression alongside immune checkpoint markers (PD-1, PD-L1, CTLA-4) and tumor-infiltrating lymphocytes. This approach can reveal complex relationships between PAIP2 expression and the tumor immune microenvironment.

• Spatial transcriptomics integration: Combining PAIP2 antibody-based protein detection with spatial transcriptomics can provide insights into how PAIP2 expression patterns relate to immune gene signatures within the tumor microenvironment.

• Ex vivo drug response assays: PAIP2 antibodies can be used to monitor changes in PAIP2 expression in patient-derived tumor samples treated with immunotherapeutic agents ex vivo, potentially identifying dynamic biomarkers of response.

• Circulating tumor cell (CTC) analysis: PAIP2 antibodies could be utilized to detect PAIP2 expression in CTCs, potentially providing a liquid biopsy approach to monitoring immunotherapy response.

Recent research has demonstrated that PAIP2 is significantly negatively correlated with the gene expression levels of 14 immune checkpoints, while positively correlated with 8 others . This complex relationship suggests that PAIP2 may serve as a surrogate marker for immunotherapy response prediction, warranting further investigation using well-characterized PAIP2 antibodies.

How can researchers explore the mechanistic role of PAIP2 in mRNA deadenylation using PAIP2 antibodies?

PAIP2 antibodies can facilitate innovative approaches to investigate PAIP2's proposed role in mRNA deadenylation, particularly its function in displacing PABPC1 at the final stages when poly(A) tails are partially degraded :

• In vitro deadenylation assays: Researchers can use purified components including recombinant deadenylases, PABPC1, and synthetic RNA substrates with varying poly(A) tail lengths. Adding or immunodepleting PAIP2 (using PAIP2 antibodies) can reveal its direct effects on deadenylation rates.

• Single-molecule visualization techniques: PAIP2 antibodies conjugated to fluorophores can be used in conjunction with labeled PABPC1 and mRNA to directly visualize the dynamics of PAIP2-mediated displacement of PABPC1 from shortened poly(A) tails at the single-molecule level.

• PAIP2-PABPC1-mRNA ternary complex analysis: PAIP2 antibodies can be employed in sequential immunoprecipitation experiments to isolate and characterize the composition of mRNPs containing both PAIP2 and PABPC1, with particular attention to poly(A) tail lengths.

• Cellular deadenylation kinetics: Using PAIP2 antibodies in PAIP2 knockdown/rescue experiments, researchers can monitor how modulating PAIP2 levels affects the kinetics of poly(A) tail shortening for specific transcripts using techniques like TAIL-seq or PAL-seq.

• Structure-function analysis: PAIP2 antibodies recognizing different epitopes can be used to map functional domains involved in the proposed PABPC1 displacement activity, potentially identifying critical regions beyond the known PAM2 motif.

The hypothesis that "Paip2 does not actively compete with poly(A) for PABPC1, but instead enhances deadenylation at a later stage by promoting dissociation of PABPC1 from the shortened mRNA" could be tested by analyzing how PAIP2's affinity for PABPC1 changes as a function of poly(A) tail length, with PAIP2 antibodies being crucial for detecting these interactions under varying conditions.

How can PAIP2 antibodies be utilized in systems biology approaches to understand translational regulation networks?

PAIP2 antibodies can be integrated into systems biology frameworks to provide a more comprehensive understanding of translational regulation networks:

• Protein-protein interaction mapping: PAIP2 antibodies can be used for immunoprecipitation followed by mass spectrometry (IP-MS) to identify the complete interactome of PAIP2 under various cellular conditions. This can reveal novel interaction partners beyond the well-characterized PABPC1 binding .

• Multi-omics integration: Combining PAIP2 antibody-based proteomics with transcriptomics and ribosome profiling can provide insights into how PAIP2-mediated translation regulation affects specific mRNA subsets. This integrative approach can identify regulatory principles governing which mRNAs are most sensitive to PAIP2-mediated repression.

• Dynamic network analysis: PAIP2 antibodies can be used to monitor temporal changes in PAIP2 expression and interactions during cellular processes like differentiation, stress response, or cell cycle progression, revealing the dynamic nature of PAIP2-mediated regulation.

• Pathway enrichment analysis: GO enrichment analysis has revealed that PAIP2 is involved in diverse biological processes including humoral immune response, cell recognition, and B cell receptor signaling pathway . PAIP2 antibodies can be used to validate and further characterize these pathway connections through co-immunoprecipitation with key pathway components.

• Computational model validation: Mathematical models of translation regulation can be validated using quantitative data obtained with PAIP2 antibodies, such as absolute protein abundance measurements and binding kinetics parameters.

The complex role of PAIP2 in cellular pathways is highlighted by GSEA results showing that the B cell receptor signaling pathway is active in the PAIP2 high expression group, while processes related to cilium, cornification, immune response, and immunoglobulin complex are active in the PAIP2 low expression group . PAIP2 antibodies can help validate these computational predictions through targeted protein detection in relevant cellular systems.

What are the considerations for using PAIP2 antibodies in studying post-translational modifications of PAIP2?

Studying post-translational modifications (PTMs) of PAIP2 using antibodies requires careful consideration of several technical and experimental factors:

• Modification-specific antibodies: Consider developing or obtaining antibodies that specifically recognize modified forms of PAIP2, such as phosphorylated, ubiquitinated, or SUMOylated variants. These would complement general PAIP2 antibodies for comparative analyses.

• Sample preparation for PTM preservation:

  • Include appropriate phosphatase inhibitors (for phosphorylation studies)

  • Add deubiquitinase inhibitors like N-ethylmaleimide (for ubiquitination studies)

  • Use SUMO protease inhibitors (for SUMOylation studies)

  • Consider rapid lysis methods to minimize PTM loss during sample preparation

• Electrophoretic techniques:

  • Use Phos-tag gels to separate phosphorylated forms of PAIP2

  • Employ gradient gels (6-15%) to resolve higher molecular weight modified species

  • Consider native gel electrophoresis to preserve some labile modifications

• Enrichment strategies:

  • Perform immunoprecipitation with general PAIP2 antibodies followed by detection with PTM-specific antibodies

  • Use tandem affinity purification approaches when studying multiple modifications

  • Consider enrichment of specific PTMs (e.g., TiO2 for phosphopeptides) prior to analysis

• Mass spectrometry validation:

  • Use PAIP2 antibodies for immunoprecipitation followed by mass spectrometry to identify exact modification sites

  • Implement parallel reaction monitoring (PRM) for targeted quantification of modified PAIP2 peptides

• Functional correlation studies:

  • Correlate observed PTMs with PAIP2's interaction with PABPC1 and other partners

  • Investigate how modifications affect PAIP2's stability and half-life

  • Examine how modifications influence PAIP2's subcellular localization

The observed molecular weight discrepancy between calculated (15 kDa) and observed (26 kDa) PAIP2 strongly suggests the presence of significant post-translational modifications, making this an important area for further investigation.

How might PAIP2 antibodies facilitate investigation of PAIP2's role in RNA degradation pathways?

PAIP2 antibodies can enable several innovative experimental approaches to elucidate PAIP2's emerging role in RNA degradation pathways:

• Compositional analysis of RNA decay complexes: PAIP2 antibodies can be used for immunoprecipitation of endogenous PAIP2-containing complexes followed by RNA-seq and proteomics to identify associated mRNAs and protein factors involved in degradation processes. GSEA results suggest that RNA degradation pathways are active in PAIP2 high expression groups .

• Co-localization with P-bodies and stress granules: Immunofluorescence using PAIP2 antibodies in combination with markers for RNA processing bodies can reveal whether PAIP2 associates with these specialized cytoplasmic structures during various cellular conditions. This could provide insights into PAIP2's spatial regulation during mRNA decay.

• PAIP2-deadenylase interactions: PAIP2 antibodies can be used to investigate potential interactions between PAIP2 and deadenylase complexes (CCR4-NOT, PAN2-PAN3). Research suggests that PAIP2 may enhance deadenylation at later stages by promoting PABPC1 dissociation from shortened mRNAs .

• Temporal dynamics during mRNA decay: PAIP2 antibodies can be used in time-course experiments to track how PAIP2 association with mRNPs changes during progressive deadenylation and decay, potentially revealing stage-specific roles.

• Microenvironment-dependent regulation: PAIP2 antibodies can help investigate how factors in the tumor microenvironment affect PAIP2's role in RNA degradation, given PAIP2's correlation with immune infiltration in cancer contexts .

• Alternative polyadenylation effects: PAIP2 antibodies can be used to examine whether PAIP2 differentially regulates mRNAs with different poly(A) tail lengths or structures resulting from alternative polyadenylation.

The finding that knockdown of PAIP2 reduced microRNA-mediated silencing in luciferase reporter assays suggests that PAIP2 may play roles in multiple RNA degradation pathways, including microRNA-directed decay. PAIP2 antibodies will be essential tools for dissecting these complex regulatory mechanisms.

What are the emerging applications of PAIP2 antibodies in studying the relationship between PAIP2 and the tumor immune microenvironment?

PAIP2 antibodies offer powerful tools for investigating the newly discovered relationships between PAIP2 and the tumor immune microenvironment:

• Spatial immune profiling: Multiplex immunohistochemistry using PAIP2 antibodies in combination with immune cell markers can map the spatial relationship between PAIP2-expressing tumor cells and infiltrating immune cells. Bioinformatic analyses have revealed significant correlations between PAIP2 expression and various immune cell populations, including positive correlations with M2 macrophages and resting mast cells, and negative correlations with follicular helper T cells, CD8+ T cells, and activated NK cells .

• Single-cell analysis: PAIP2 antibodies can be adapted for cytometry by time-of-flight (CyTOF) or imaging mass cytometry to simultaneously detect PAIP2 and immune markers at single-cell resolution, revealing heterogeneity within tumor samples.

• Functional immunomodulation studies: PAIP2 antibodies can be used to monitor changes in PAIP2 expression following treatment with immunomodulatory agents, potentially uncovering mechanisms of resistance or sensitivity to immunotherapy.

• Experimental model validation: PAIP2 antibodies can help validate findings from computational analyses in experimental models by confirming protein-level correlations between PAIP2 and immune parameters. Analysis using the ESTIMATE algorithm has shown that StromalScore, ImmuneScore, and ESTIMATEScore are significantly lower in PAIP2 high expression groups compared to PAIP2 low expression groups .

• Extracellular vesicle (EV) analysis: PAIP2 antibodies can be used to investigate whether PAIP2 is present in tumor-derived EVs and how this might influence immune cell function in the tumor microenvironment.

• Checkpoint inhibitor response prediction: PAIP2 antibodies can facilitate the development of immunohistochemistry-based assays to predict response to immune checkpoint inhibitors, given that PAIP2 expression negatively correlates with IPS (Immunophenoscore) data, suggesting patients with low PAIP2 expression may have better responses to immune checkpoint inhibitors .